Chitin is a long-chain polysaccharide of β-(1-4)-linked N-acetyl-D-glucosamine units and is found in many places throughout the natural world. It is the main component of the cell walls of fungi, the exoskeletons of arthropods such as crustaceans (e.g., crabs, lobsters and shrimps (including Pandalus Borealis)) and insects, the radulas of mollusks, and the beaks of cephalopods, including squid and octopuses. It is the second most common natural polysaccharide with an annual world production estimated at 2.3×109 tons (see Biopolymers, Vol. 6: Polysaccharides II: Polysaccharides from Eukaryotes, Vandamme E. J., De Baets S., Steinbüchel A., Wiley-VCH, New York, 2002, p. 488.).

Chitosan, a poly[β-(1→4)-2-amino-2-deoxy-D-glucopyranose], is a biodegradable, biocompatible and non-toxic linear aminopolysaccharide obtained by deacetylating chitin. Full deacetylation of chitin produces chitosan with a degree of deacetylation (DD) of 100% and comprised only of 3-(1-4)-linked D-glucosamine (deacetylated units):

Chitosan with degrees of deacetylation (DD) smaller than 100% further comprises some original N-acetyl-D-glucosamine units. The degree of deacetylation of commercially available chitosan is usually in the range of 50-100%.
Chitosan is one of a few biopolymers that is cationic when protonated. Cationic polymers can generally adsorb on the cell walls of bacteria and thus act as antibacterials. For example, many studies outline the efficacy of chitosan in wound treatment. Utilization of chitosan in other applications has however been limited due to its insolubility in water when in neutral form. Its solubility is limited to diluted aqueous acid solution of pH<6.5.
N,N,N-trimethylchitosan (TMC) is a polycation generally obtained by methylating chitosan. It has the following ideal chemical formula:
with a counterion to balance the electrical charge.
However, such an “ideal” TMC has never been produced. In fact, TMC reported in the literature has low degrees of quaternization (DQ) of the nitrogen atom (30-40% at most), which means that at least some D-glucosamine units only bear zero, one or two methyl groups on their nitrogen atom, rather than bearing three of them.
Typically, methods of producing TMC comprise carrying out successive methylation reactions on chitosan, using iodomethane (methyl iodide) or dimethylsulfate in the presence of a base, often sodium hydroxide, under various experimental conditions (see Curti E., Britto D., Campana-Filho S. P., Macromol. Biosci., 2003, 3, 571-576 and Hamman J. H., Kotzé A. F., Drug Dev. Ind. Pharm., 2001, 27, 373-380). However, methylation of chitosan under these conditions leads to the formation of a mixture of unmethylated, mono, di and trimethylated amines.
Indeed, chemical modification of polysaccharides presents many difficulties such as a lack of solubility of the polysaccharides in many organic and inorganic solvents (including water), the presence of various other chemical functions (thus the modification must be regioselective) and the high number of repeating units. Nevertheless, the synthesis of TMC has been the subject of many scholarly papers as it provides a polycationic chitosan derivative that is soluble in water. The formation of TMC indeed introduces positive charges and thus provides a water-soluble product in a wide pH range even when partially quaternized. It is thus desirable to produce TMC having a high degree of quaternization.
It has further been reported that attempts to increase the degree of quaternization to higher than 30-40% result in methylation of the oxygen atoms of the OH groups at position 3 and/or position 6 to an extent, which is referred to as the degree of O-substitution (O-DS). High degrees of O-substitution lead to a decrease in the TMC solubility in water as hydrophilic —OH groups are replaced by more hydrophobic—OCH3 groups. It is thus desirable to produce TMC having a low degree of O-substitution.
Chitosan having high degrees of quaternization (DQ) on chitosan has been obtained (DQ of 90.5%) in a process involving three methylation steps, using iodomethane and sodium hydroxide; however, these conditions also lead to high degrees of O-substitution, particularly at positions 3 and 6 (O-3 and O-6 methylation), respectively of 82.8 and 98.5% (Polnok A., et al., Eur. J. Pharm. Biopharm., 2004, 57, 77-83). The formation of methyl ether (O-methylation) by substitution of alcohol groups undesirably leads to a decreasing solubility of chitosan derivatives and possibly to a water insoluble product (Curti E., Britto D., Campana-Filho S. P., Macromol. Biosci., 2003, 3, 571-576, Polnok A., et al., Eur. J. Pharm. Biopharm., 2004, 57, 77-83, and Snyman D., Hamman J. H., Kotze J. S., Rollings J. E., Kotzé A. F., Carbohydr. Polym., 2002, 50, 145-150).
Moreover, the use of sodium hydroxide or a strong alkaline environment decreases the molecular weight of the polymer. With experimental conditions that prevent O-methylation, low degrees of quaternization have generally been obtained (Polnok A., et al., Eur. J. Pharm. Biopharm., 2004, 57, 77-83).
Alternatively, when N,N-dimethylaminopyridine was used as a base instead of sodium hydroxide to avoid degradation of chitosan, low DQs (7.3-9.6%) were obtained (Hamman J. H., Kotzé A. F., Drug Dev. Ind. Pharm., 2001, 27, 373-380).
TMC has also been synthesized using dimethylsulfate as the methylating agent (Britto D., Forato L. A., Assis O. B. G., Carbohydr. Polym., 2008, 74, 86-91, Britto D., Assis B. G. O., Carbohydr. Polym., 2007, 69, 305, and Britto D., Assis B. G. O., Intl. J. Biol. Macromol., 2007, 41, 198).
Chitosan has also been N-permethylated by reaction with formaldehyde followed with sodium borohydride. The N-permethylated chitosan was reacted with iodomethane to obtain TMC. The resulting TMC iodide has a high degree of O-substitution of 60% and exhibits antibiotic activity, but is insoluble in water (Muzzarelli R. A. A., Tanfani F., Carbohydr. Polym., 1985, 5, 297).
Preparation of mixed N,N,N-trialkylchitosan derivatives has also been reported with different alkyl iodide (Avadi M. R., Zohuriaan-Mehr M. J., Younessi P., Amini M., Rafiee Tehrani M., Shafiee A., J. Bioact. Compat. Polym., 2003, 18, 469 and Bayat A., Sadeghi A. M. M., Avadi M. R., Amini M., Rafiee-Tehrani M., Shafiee A., Majlesi R., Junginger H. E., J. Bioact. Compat. Polym., 2006, 21, 433).
TMC has drawn interest because of its multiple uses. Known uses of TMC include for example:
increasing the absorption of molecules through mucosae (for example through the intestine wall),
controlled release of various substances including genes and proteins, and
use as an antimicrobial agent.
N,N,N-trimethylchitosan has an increased density of positive charge (compared to chitosan) and it has been shown to open the tight junctions of epithelial cells. It has thus been proven to be a potent intestinal absorption enhancer for hydrophilic and macromolecular drugs in physiological pH (Thanou M., Florea B. I., Langemeyer M. W. E., Verhoef J. C., Junginger H. E., Pharm. Res., 2000, 17, 27; Thanou M., Verhoef J. C., Verheijden J. H. M., Junginger H. E., Pharm. Res., 2001, 18, 823; Thanou M., Kotzé A. F., Scharringhausen T., Leuben H. L., De Boer A. G., Verhoef J. C., Junginger H. E., J. Controlled Release, 2000, 64, 15; Thanou M., Verhoef J. C., Junginger H. E., Adv. Drug Delivery Rev., 2001, 52, 117; and Florea B. I., Thanou M., Junginger H. E., Borchard G., J. Control. Release, 2006, 110, 353). The density of positive charge is known to have an important effect on drug absorption enhancing properties (Kotzé A. F., LueEn H. L., Leeuw B. J., Boer B. G., Verhoef J. C., Junginger H. E., Pharmaceutical Research, 1997, 14, 1197; and Kotzé A. F., Thanou M., Lueben H. L., de Boer A. G., Verhoef J. C., Junginger H. E., Eur. J. Pharm. Biopharm., 1999, 47, 269). It has been reported that the higher the degree of quaternization, the greater the increase in absorption.
According to J. Pharm. Sci., 2008, 97(5), 1652-1680, polymers increasing transepithelial penetration are polycations (chitosan, poly-L-arginine (poly-L-Arg), aminated gelatin), polyanions (N-carboxymethylchitosan, poly (acrylic acid)) and thiolated polymers (carboxymethyl cysteine-cellulose, polycarbophil (PCP)-cysteine, chitosan-thiobutylamidine, chitosan-thioglycolic acid, chitosan-glutathione conjugate).
TMC has been used in certain other applications such as in gene delivery, in colonic drug delivery and as an antibacterial (Rünarsson Ö. V., et al., Eur. Polym. J., 2007, 43, 2660-2671; Borchard G., Adv. Drug Delivery Rev., 2001, 52, 145; Dodou D., Breedveld P., Wiering a P. A., Euro. J. Pharm. Biopharm., 2005, 60, 1; Kim C. H., Choi J. W., Chun H. J., Choi K. S., Polym. Bull., 1997, 38, 387; Kean T., Roth S., Thanou M., J. Control. Release, 2005, 103, 643; and Jia Z., Shen D., Xu W., Carbohydr. Res., 2001, 333, 1). A structure-activity relationship study reveals that N-quaternization on chitosan and on chitooligomers was responsible of the antibacterial activity against Staphylococcus aureus at pH 7.2 (Rünarsson Ö. V., et al., Eur. Polym. J., 2007, 43, 2660-2671).
Cationic polymers, including protonated chitosan, are known to possess antibacterial properties (Lim, S. H. et al. J. Macromol. Sci.-Polym. Rev., 2003, C43, 223.). Factors affecting antibacterial activity of polymers generally are the structure, the molecular weight, the nature of the counterions, and the hydrophobicity (Kenawy, E. et al. Biomacromolecules, 2007, 8, 1359-1384). The killing time can be as low as 30 min. for chitosan (EI-Sharif, A. A. et al. Curr. Microbiol., 2011, 62, 739-745).
Additionally, hydrophilic biopolymers generally are known to have an emollient effect.
Accordingly, there thus remains a need for improved methods of producing N,N,N-trialkylaminopolymers, particularly N,N,N-trialkylaminopolysaccharides that have high degrees of quaternization and low degrees of other heteroatom-substitution. Particularly, there is a need for improved methods of producing N,N,N-trialkylchitosan having a high degree of quaternization and a low degree of O-substitution.